The ultimate goal of the proposed research is the definition of genes responsible for congenital heart defects in Down syndrome. The proposed work represents a joint effort to narrow the region responsible for congenital heart defects in Down syndrome and then to isolate genes expressed from that region in an embryonic heart cDNA library. We have defined a small region (3-5 mb) of chromosome 21 likely to contain the gene(s) for DS-CHD. Based on a clue that both heart and lung defects in Down syndrome may result from the same tendency of Down syndrome fetal heart and lung fibroblasts to aggregate avidly, we will also prepare and screen cDNA libraries from trisomy 21 fetal heart and lung fibroblasts that show abnormal adhesion as well as from an embryonic lung cDNA library. The expression of candidate sequences by in situ hybridization in the developing endocardial cushions and/or pulmonary acinar cells will be utilized to determine candidate genes that could mediate the heart and lung defects in Down syndrome. If linked to chromosome 21, autosomal dominant pedigrees with endocardial cushion defects (ECD) will also be investigated and used to narrow the DS-CHD region. Thus, in this grant, we propose to: 1) Use cases involving partial aneusomy for chromosome 21 to further decrease the size of the DS-CHD region. In addition, we will utilize the trisomy 16 mouse that also develops endocardial cushion defects to delimit the size of the segment of chromosome 21 pathogenic for CHD. 2) Construct human embryonic heart and lung and trisomy 21 fetal cardiac and pulmonary fibroblast libraries as these represent the target tissues for cardiopulmonary defects in Down syndrome. 3) Screen these libraries and isolate genes by a novel recombination- based assay (RBA) we developed for this purpose. Portions of genes in this subregion will be identified by other methodologies such as computer analyses, exon amplification or hybrid selection and these sequences will be placed in our R6K supF plasmid, pMAD3. The RBA will then be performed both to confirm the tissue and time of expression and to isolate a longer gene fragment. 4) In situ hybridization of candidate genes will be performed to detect messages expressed in the endocardial cushions and/or acinar lung cells during organogenesis. Thus a gene "map" will be elaborated for sequences in a particular region of chromosome 21 known to be involved in abnormal cardiogenesis. At the least, this map will serve as a guideline for future studies to couple a "genic" initiative to characterize the time and tissue of transcription of particular regions to the "genomic" initiative. The efficient accomplishment of such a "genic" initiative will increase the value of the "genomic" initiative.